Imaging agents, precursors thereof and methods of manufacture

ABSTRACT

Compounds of formula (I) and (II):wherein groups R1, R2, RE, PG1 and PG2 have the definitions provided in the specification, methods of manufacture and methods of use.

BACKGROUND OF THE INVENTION

Short half-lived radioactive compounds are useful as imaging agents to diagnose cancers and abscesses. In the routine production of such imaging agents, safety and production considerations must be taken into account. It is advantageous to minimize contact time while ensuring quick and consistent production of the imaging agents.

There are generally two types of imaging procedures conducted. In positron emission tomography (PET), two beta rays emitted from the decaying radionuclide are detected. In single photon emission computed tomography (SPECT), one beta ray emitted from the decaying radionuclide is detected. PET provides a more exact location of tumors. However, SPECT is simpler and easier to use, and is therefore used more often.

Generally speaking, PET uses radio-compounds labeled with the positron-emitters such as ¹⁸F, ¹¹C, ¹³N and ¹⁵O, SPECT uses radio compounds labeled with the single-photon-emitters such as ¹⁸F, ¹¹C, ¹³N and ¹⁵O, although ⁷⁵Br and ¹²⁴I can also be used. SPECT, on the other hand, generally uses radionuclides that have more neutrons than protons, such as ⁶⁷Ga, ⁷⁷Br, ¹²³I, ¹²⁴I, ¹²⁵I, ¹²⁶I, ¹³¹I and ²⁰¹TI.

In the art, glucose-based and amino acid-based compounds have been used as imaging agents. Amino acid-based compounds are more useful in analyzing tumor cells, due to their faster uptake and incorporation into protein synthesis. Of the amino acid-based compounds, ¹¹C- and ¹⁸F-containing compounds have been used with success. ¹¹C-containing radiolabeled amino acids suitable for imaging include, for example, L-[1-¹¹C]leucine (Keen et al. J. Cereb. Blood Flow Metab. 1989 (9):429-45), L-[1-¹¹C]tyrosine (Wiesel et al. J. Nucl. Med. 1991 (32):2041-49), L-[methyl-¹¹C]methionine (Comar et al. Eur. J. Nucl. Med. 1976 (1):11-14) and L-[1-¹¹C]methionine (Bolster et al. Appl. Radiat. Isot. 1986 (37):1069-70). However, ¹¹C-containing radiolabeled amino acids have limited applicability, due to the short half-life of ¹¹C (T_(1/2) =20 min.). Therefore, great effort was directed toward the synthesis and evaluation of other radionuclides, such as those listed above. Two ¹⁸F-containing radiolabeled amino acids, 4-[¹⁸F]fluoro-L-phenylalanine and 2-[¹⁸F]fluoro-L-tyrosine, exhibit protein incorporation, but can only be synthesized with uncorrected yields less than 5%. See Arnstein et al. Biochem. J. 1984 (1):340-46 and Coenen et al. Radioisot. Klinik. Forschung 1988 (18):402-40.

More recently, position 2- and 3-L-[¹⁸F]fluoro-α-methyl tyrosine have been synthesized by electrophilic substitution (Tomiyoshi et al. Nucl. Med. Commun. 1997 (18):169-75). Unfortunately, the radiochemical yield from such a production is low. Another more recent compound to be synthesized is o-(2-[¹⁸F]fluoroethyl)-L-tyrosine (Wester et al. J. Nucl. Med. 1999 (40):205-212). This paper describes the synthesis of the target compound, using a nucleophilic reaction, in a 40-60% yield. However, the reaction requires a two-step synthesis.

SUMMARY OF THE INVENTION

An object of the present invention is to develop new imaging agents suitable for use in PET and SPECT and which overcome the disadvantages of known compounds.

Another object of the present invention is to provide a method of manufacturing such compounds via the nucleophilic route using a one-step synthesis.

A further object of the invention is to provide precursors useful for manufacturing the new imaging agents of the invention.

These objects are achieved by the present invention.

The present invention includes compounds of formula (I), or pharmaceutically acceptable salts thereof:

wherein

the C marked with an asterisk represents a chiral center and the compound is present in the L-form, the D-form or as a racemic mixture;

R₁ is selected from the group consisting of a single bond, phenyl, and a group of formula (a), (b), (c) or (d)

The present invention also includes compounds of formula (II), which are useful for preparing the compounds of formula (I):

wherein

R₁ is the same as indicated for the compounds of formula (I);

R₂ is H or a group —R₃—O—R₄, wherein R₃ is C₁-C₇ alkyl and R₄ is H or a leaving group, preferably a sulfonyl group such as tosyl, trifyl, mesyl, trimsyl, tripsyl, brosyl or nosyl;

PG₁ is a carboxyl protecting group; and

PG₂ is an amino protecting group.

The present invention also includes a method of synthesizing compounds of formula (I) by reacting a compound of formula (IIc):

wherein R₁, R₂, PG₁ and PG₂ are the same as indicated for the compounds of formula (I), and R₃ is a leaving group, preferably a sulfonyl group such as tosyl, trifyl, mesyl, trimsyl, tripsyl, brosyl or nosyl,

with a salt of RE, where RE is the same as indicated for the compounds of formula I, to produce a compound of formula (Ia):

wherein R₁, R₂, RE, PG₁ and PG₂ are the same as above; and removing the protecting groups.

In this invention, compounds of formula I have been developed:

wherein

the C marked with an asterisk represents a chiral center and the compound is present in the L-form, the D-form or as a racemic mixture; R₁ is selected from the group consisting of a single bond, phenyl, and a group of formula (a), (b), (c) or (d)

R₂ is C₁-C₇ alkyl; and

RE is selected from the group consisting of ¹¹C, ¹³N, ¹⁵O, ¹⁸F, ⁶⁷Ga, ⁷⁵Br, ⁷⁷Br, ¹²³I, ¹²⁴I, ¹²⁵I, ¹²⁶I, ¹³¹I, and ²⁰¹TI, preferably ¹²³I, ¹²⁵I, and ¹⁸F.

Preferably, R₁ is phenyl. When R₁ is phenyl, the —O—R₂—RE group can be para, meta or ortho the CH₂ group. It is preferred that the —O—R₂—RE group is para the CH₂ group.

R₂ is preferably C₂-C₆ alkyl, more preferably C₂-C₅ alkyl. It is most preferable that R₂ is propyl.

The compounds of formula (I) contain a chiral center at the α-C. The compounds of the invention can therefore be present in the L-form, the D-form, or as a racemic mixture. It is preferred that the compounds be present in the L-form.

The present invention also includes compounds of formula (II):

wherein

R₁ is the same as indicated for the compounds of formula (I);

R₂ is H or a group —R₃—O—R₄, wherein R₃ is C₁-C₇ alkyl and R₄ is H or a leaving group, preferably a sulfonyl group such as tosyl, trifyl, mesyl, trimsyl, tripsyl, brosyl or nosyl;

PG₁ is a carboxyl protecting group; and

PG₂ is an amino protecting group.

The compounds of formula (II) are useful as intermediates in the preparation of compounds of formula (I).

More specifically, the compounds of formula (II) may be divided into subgroups (IIa), (IIb) and (IIc):

which are important intermediates in the preparation of compounds of formula (I),

wherein in each case

R₁ and R₂ are the same as indicated for the compounds of formula (I);

R₃ is a leaving group, preferably a sulfonyl group such as tosyl, trifyl, mesyl, trimsyl, tripsyl, brosyl or nosyl;

PG₁ is a carboxyl protecting group; and

PG₂ is an amino protecting group.

As noted above, the leaving groups for compounds of formula (II) are preferably sulfonyl groups, however any suitable leaving group can be used, as known to those of ordinary skill in the art. Especially preferred are tosyl, trifyl, mesyl, trimsyl, tripsyl, brosyl or nosyl, with tosyl, trifyl and mesyl being more preferred and tosyl being most preferred.

The protecting groups (PG₁ and PG₂) can be any suitable carboxyl protecting group and amino protecting group, as known to those of ordinary skill in the art. Reference is made to “Protective Groups in Organic Chemistry,” ed. J. F. W. McOmie, Plenum Press, 1973; and T. W. Greene & P. G. M. Wuts, “Protective Groups in Organic Synthesis,” John Wiley & Sons, 1991, each incorporated by reference. PG₁ may be, for example, an alkyl group such as methyl, ethyl or propyl. PG₂ may be, for example, a Boc group.

Generally, the reaction scheme for preparing compounds of formula (I) is as follows. A starting compound of formula (III):

wherein R₁ and PG₁ are the same as indicated for the compounds if formula (I), such as α-methyl tyrosine C₁-C₃ alkyl ester, α-methyl serine C₁-C₃ alkyl ester or α-methyl hydroxy-tryptophan C₁-C₃ alkyl ester,

is reacted in a suitable solvent with a compound providing a suitable protecting group PG₂, such as di-t-butyl dicarbonate (which provides a Boc group), to protect the amine, giving the compound of formula (IIa).

The compound of formula (IIa) is thereafter reacted in a suitable solvent with a halogenated C₁-C₇ alkanol, to give the compound of formula (IIb).

The compound of formula (IIb) is thereafter reacted in a suitable solvent with a halogenated leaving group, such as halogen-tosyl, to give the compound of formula (IIc).

The compound of formula (IIc) is reacted in a suitable solvent with a salt of RE, as defined above, such as K-¹⁸F, to give the compound of formula (Ia). The compound of formula (Ia) is converted into the compound of formula (I) by removing the protecting groups (PG₁ and PG₂), for example by using TFA.

In the exemplified embodiment, 3-[¹⁸F]fluoropropyl-α-methyl tyrosine, the radiochemical yield for the compound of the invention was much higher than ¹¹C- and ¹⁸F-labeled amino acids synthesized using an electrophilic process. In addition, the compounds of the invention are stable and show high uptake in tumors. The compounds of the invention are useful to differentiate the degree of malignancy of tumors (e.g., brain, breast, prostate, colon, lung, liver, pancreas, gastric, lymphoma, uterine, cervical, extremitis, sarcoma and melanoma). The compounds of the invention are also useful to image neurological disorders (e.g. Alzheimer's, Huntington's), abscess, inflammation and infectious diseases. Suitable dosages are known or could be easily determined by those of ordinary skill in the art.

EXAMPLES Experiment 1 Synthesis of N-t-Butyloxycarbonyl-α-methyltyrosine methylester

α-Methyltyrosine HCl methylester MW 245.7 Wt 2.54 g mmol 10.33 Di-t-butyl dicarbonate MW 218.25 Wt 2.20 g mmol 10.08 Triethylamine MW 101 Wt 2.50 g or 3.6 ml mmol 24.8

The α-methyltyrosine methylester (2.54 g) was dissolved in 16 ml of dimethylformamide (DMF) which became cloudy with the addition of triethylamine (3.6 ml). Di-t-butyl dicarbonate (2.20 g) was added and the mixture stirred overnight. After evaporation of DMF under high vacuum the material was treated with ether (30 ml) which produced a precipitate. This precipitate was filtered and discarded. Evaporation of the filtrate yielded 3.38 g (dried under vacuum) of a colorless liquid. TLC analysis using Hexane. Ethyl Acetate (1:1) showed the product at Rf 0.7 and the starting material at 0.1.

Experiment 2 Synthesis of N-t-Butyloxycarbonyl-O-[3-hydroxypropyl]-αmethyltyrosine methylester

N-t-Butyloxycarbonyl-α-methyltyrosine MW 309.2 methyl ester Wt 1.7 g mmol 5.50 3-Bromopropanol MW 138.9 Wt 924 mg or 0.6 ml mmol 6.65 Sodium ethoxide (NaOEt) MW 68.05 Wt 742 mg mmol 10.90

The N-t-butyloxycarbonyl-α-methyltyrosine methylester (1.70 g) from the previous synthesis was dissolved in 25 ml of absolute ethanol and treated with dry sodiun ethoxide (742 mg). 3-Bromopropanol (924 mg, 0.6 ml) was added and the solution was refluxed for 5 hours. The ethanol was then evaporated, 100 ml of dichloromethane added and the solution washed with 100 ml of water. After drying over MgSO₄ the solvent was evaporated to yield two spots on a TLC plate developed in EtOAc:Hexane 1:1. The first spot was product and the second was starting material. The product was purified on a silica gel column using EtOAc:Hexane (1:1) A yield of 620 mg (MW 367.2, 31%) was obtained.

Experiment 3 Synthesis of N-t-Butyloxycarbonyl-O-[3-tosylpropyl]-α-methyltyrosine methylester

N-t-Butyloxycarbonyl-O-[3-hydroxypropyl]-α- MW 367.2 methyltyrosine Methylester Wt 620 mg mmol 1.69 p-Toluenesulfonyl chloride (tosyl chloride) MW 190.5 Wt 400 mg mmol 2.10 mg Pyridine MW 79 d = 0.98 g/ml Wt 500 mg mmol 6.33

The N-t-butyloxycarbonyl-O-[3-hydroxypropyl]-α-methyltyrosine methylester (620 mg) from the previous synthesis was dissolved in 10 ml of chloroform and carefully treated with p-toluenesulfonyl chloride (400 mg, which is tosyl chloride) and 0.5 ml of pyridine (500 mg). After allowing to react overnight the volume of solvent was reduced to 2 ml and added to a silica column for purification. The column was washed with EtOAc:Hexane 2:1 and the fractions #4-6 were combine to yield 400 mg, 45.4% of product. Analysis by TLC developed in EtOAc:hexane showed the product between the starting material and tosyl chloride.

Experiment 4 Synthesis of 3-¹⁸F-fluoropropyl-α-methyltyrosine ([¹⁸F]FPAMT)

Fluorine-18 in 1.0 milliliter of 95% oxygen-18 enriched water was transferred from the cyclotron target to an anion exchange column (in the OH form) where the ¹⁸F was trapped, and the ¹⁸O-water passed through and was collected in a bottle. The ¹⁸F was washed from the column with 1.5 milliliters of 0.01 molar potassium carbonate into a reaction vessel containing 26 mg of 2,2,2-Kryptofix. The resulting potassium fluoride/Kryptofix complex was subsequently dried, first by distillation under vacuum and then by azeotropic distillation of the remaining water with the addition of three 1.5 milliliter portions of acetonitrile.

The ¹⁸F complex was allowed to exchange with the tosyl group on 4 milligrams of N-t-butyloxycarbonyl-O-[3-tosylpropyl]-α-methyltyrosine methylester (tosyl precursor; PAMT-OTs) which was dissolved in 0.5 milliliters of acetonitrile (tube #1). This exchange took 15 minutes at 95° C.

The cooled acetonitrile solution was passed onto a dry normal 500 milligram silica column (Sep-Pak column (Not C-18)). This material was eluted (washed from the column) with 3 milliliters of diethyl ether into tube #2 where the solvents (ether/acetonitrile) were evaporated to about 1 milliliter.

Addition of 0.1 milliliter of trifluoroacetic acid for 20 minutes at room temperature was used to remove the protecting group. The solution was evaporated to dryness and 2 milliliters of water were added to this tube (#2). After warming (40° C.), the water solution was passed through a 0.22 micron filter for sterilization.

The final product was analyzed for sterility and by High Performance Liquid Chromatography and TLC for radiochemical purity.

Experiment 5 In Vitro Cellular Uptake Assay

Each well containing 50,000 breast cancer cells, cell line 13762 (0.5 ml/well) was added with 2 μCi of ¹⁸F-FDG and ¹⁸F-FPAMT. After 0.5-4 hours incubation, the cells were washed with phosphate buffered saline 3 times and followed by trypsin to loosen the cells. The cells were counted by a gamma counter.

An increased uptake of ¹⁸F-FDG and ¹⁸F-FPAMT as a function of time was observed in the breast cancer cell line. ¹⁸F-FPAMT showed slightly higher uptake than ¹⁸F-FDG at 0.5 and 2 hours incubation.

Experiment 6 In Vivo Tissue Distribution

Fischer 344 tumor-bearing rats (weight 150-155 g) with tumor volume 1.2 cm were administered with ¹⁸F-FDG and ¹⁸F-FPAMT (10 μCi/rat, i.v.). Biodistribution studies were conducted at 30 min, 2 and 4 hours.

Tumor/blood and tumor/muscle ratios were increased as a function of time for both ¹⁸F-FDG and ¹⁸F-FPAMT. There was an increased bone uptake in ¹⁸F-FPAMT at 2 hours, suggesting in vivo defluorination might have occurred. Tables 1 and 2 show % of injected dose per gram of tissue weight (values shown represent the standard deviation from 3 animals).

TABLE 1 Biodistribution of ¹⁸F-FPAMT in Breast Tumor-Bearing Rats % of injected ¹⁸F-FPAMT dose per organ or tissue 30 min 2 h 4 h Blood 0.638 ± 0.021 0.298 ± 0.032 0.081 ± 0.007 Lung 4.812 ± 0.457 0.541 ± 0.084 0.105 ± 0.008 Liver 6.542 ± 1.142 3.587 ± 0.500 0.930 ± 0.049 Stomach 0.440 ± 0.052 0.190 ± 0.024 0.070 ± 0.002 Spleen 0.404 ± 0.023 0.215 ± 0.029 0.067 ± 0.009 Kidney 8.060 ± 0.101 7.721 ± 1.027 1.467 ± 0.236 Thyroid 0.730 ± 0.126 0.860 ± 0.045 1.220 ± 0.260 Muscle 0.318 ± 0.043 0.154 ± 0.017 0.055 ± 0.012 Intestine 0.838 ± 0.058 0.627 ± 0.209 0.148 ± 0.015 Tumor 0.454 ± 0.016 0.312 ± 0.024 0.069 ± 0.003 Bone 0.447 ± 0.045 1.256 ± 0.117 1.502 ± 0.214 Heart 0.445 ± 0.019 0.219 ± 0.024 0.054 ± 0.007 Tumor/Blood 0.712 ± 0.013 1.079 ± 0.171 0.853 ± 0.036 Tumor/Muscle 1.464 ± 0.138 2.057 ± 0.176 1.322 ± 0.341 Tumor/Lung 0.095 ± 0.006 0.599 ± 0.079 0.658 ± 0.081

TABLE 2 Biodistribution of ¹⁸FDG in Breast Tumor-Bearing Rats % of injected ¹⁸FDG dose per organ or tissue 30 min 2 h 4 h Blood 0.58 ± 0.044 0.47 ± 0.072 0.25 ± 0.017 Lung 0.62 ± 0.054 0.52 ± 0.137 0.23 ± 0.022 Liver 0.88 ± 0.144 0.85 ± 0.040 0.79 ± 0.039 Spleen 0.57 ± 0.068 0.45 ± 0.046 0.19 ± 0.012 Kidney 0.96 ± 0.123 0.85 ± 0.158 0.51 ± 0.037 Muscle 1.42 ± 0.087 0.69 ± 0.072 0.31 ± 0.048 Bone 0.30 ± 0.036 0.28 ± 0.013 0.22 ± 0.017 Tumor 0.90 ± 0.113 1.05 ± 0.111 0.69 ± 0.097 Tumor/Blood 1.57 ± 0.188 2.24 ± 0.204 2.41 ± 0.676 Tumor/Muscle 0.66 ± 0.066 1.52 ± 0.175 2.20 ± 0.158

The invention has been described herein with reference to certain preferred embodiments. However, as obvious variations thereon will become apparent to those skilled in the art, the invention is not to be considered as limited thereto. 

We claim:
 1. A compound of formula (I), or a pharmaceutically acceptable salt thereof:

wherein the C marked with an asterisk represents a chiral center and the compound is present in the L-form, the D-form or as a racemic mixture; R₁ is selected from the group consisting of a single bond, phenyl, and a group of formula (a), (b), (c) or (d)

R₂ is C₁-C7 alkyl; and RE is selected from the group consisting of ⁷⁵Br, ¹²⁴I and ¹⁸F.
 2. The compound of claim 1, wherein R₁ is a single bond.
 3. The compound of claim 1, wherein R₁ is phenyl.
 4. The compound of claim 3, wherein the —O—R₂—RE group is para the CH₂ group on the phenyl.
 5. The compound of claim 3, wherein the —O—R₂—RE group is meta the CH₂ group on the phenyl.
 6. The compound of claim 3, wherein the —O—R₂—RE group is ortho the CH₂ group on the phenyl.
 7. The compound of claim 1, wherein R₁ is a group of formula (a), (b), (C) or (d).
 8. The compound of claim 1, wherein R₂ is C₂-C₆ alkyl.
 9. The compound of claim 1, wherein R₂ is C₂-C₅ alkyl.
 10. The compound of claim 1, wherein the compound is present in the L-form.
 11. The compound of claim 1, wherein the compound is present in the D-form.
 12. The compound of claim 1, wherein the compound is present as a racemic mixture.
 13. The compound of claim 1, wherein the compound is 3-[¹⁸F]fluoro(C₂-C₆)-α-methyl tyrosine.
 14. The compound of claim 13, wherein the compound is 3-[¹⁸F]fluoropropyl-a-methyl tyrosine. 